These days, most amino acids are being produced using microorganisms, constructed by random mutagenesis techniques. These microbial strains have a shortcoming in that it is difficult to additionally improve the strains, because it is difficult to understand the precise physiological metabolism thereof. Thus, a rational design technique is required, in which only specific genes are deleted or amplified, such that desired amino acids are produced.
As used herein, the term “branched-chain amino acids” refers to three substances, valine, leucine and isoleucine, among nine essential amino acids. Unlike most other amino acids which are metabolized in the liver, the branched-chain amino acids are metabolized mainly in muscles, such that they are used as energy sources for moving the body.
Currently, the market share of the branched chain amino acids is only 1%, but is increasing fast, since it was recently reported that the branched chain amino acids play an important role in maintaining and building muscles for moving the body. In particular, L-valine has been used as a feed component, since it was reported that L-valine has high reducing power and serves to increase the ability of cows to produce milk. Although L-valine has generally been produced from bacterial strains, such as Brevibacterium, Corynebacterium or Serratia sp., it was recently reported that L-valine was produced in E. coli strains, which are easy to culture and allows the use of advanced molecular biological techniques. In particular, Ajinomoto Co., Inc., Japan, reported that a bacterial strain having resistance to L-valine was screened using a random mutagenesis technique and it produced 23.4 g/l of L-valine (U.S. Pat. No. 6,737,255).
Also, the production of L-valine-producing microorganisms (Corynebacterium glutamicum) using a rational design technique was recently reported, in which 130 mM of L-valine was produced using microorganisms, obtained by deleting two genes (ilvA and panB), amplifying an operon (ilvBNC) involved in L-valine biosynthesis, and then removing feedback inhibition of the ilvN gene (Veronika et al., Appl. Environ. Microbiol., 71:207, 2005). However, an example, in which L-valine was produced in E. coli using the rational design method, has not been reported yet.
Among various rational design methods, in silico simulation methods, particularly analysis and prediction techniques using metabolic pathways, have recently shown its possibilities along with rapidly increasing genome information. In particular, by the combination of microbial metabolic pathway models with mathematical models and optimization techniques, it has become possible to predict reactions in metabolic pathways, which occur after the removal or addition of genes. Also, it is known that a technique of analyzing metabolic fluxes using metabolic pathways shows ideal cellular metabolic fluxes without requiring dynamic information and can substantially simulate and predict the precise behavior of cells. Metabolic flux analysis obtains an ideal metabolic flux space that cells can reach using only a set of metabolic mass balancing reactions and cell composition information, and it aims to maximize or minimize a specific objective function through an optimization method (either maximization of cell growth rate or minimization of metabolic regulation by specific perturbation). In addition, metabolic flux analysis can be used to analyze the lethality of a specific gene in a desired metabolite through bacterial strain improvement and to understand the characteristics of metabolic pathways in bacterial strains. Moreover, studies on the various applications of metabolic flux analysis for predicting the change in metabolic pathway flux, which is caused by the removal or addition of genes, have been reported.
Thus, in the art to which the present invention pertains, there is an urgent need to develop microorganisms having high productivity of branched-chain amino acids, particularly, E. coli strains having high productivity of L-valine, using the rational design method which comprises reconstructing metabolic pathways through the deletion of specific genes and amplifying desired genes, unlike the prior random mutagenesis techniques.
Accordingly, the present inventors have made many efforts to develop microorganisms having high productivity of branched-chain amino acids, particularly L-valine, using the rational design method. As a result, the present inventors have found that mutant microorganisms, produced by substituting a native promoter containing the attenuators of L-valine biosynthesis operons with a strong promoter, inserting an operon containing the substituted promoter into a recombinant vector, and then introducing the recombinant vector into E. coli having a deletion of ilvA, leuA and panB genes involved in competitive pathways, can produce L-valine with high productivity and efficiency, thereby completing the present invention.